PROJECT SUMMARY/ABSTRACT: Metastatic melanoma is a lethal disease with a dismal 20% five-year survival. Immunotherapy and molecularly targeted treatments improve survival but responses vary across patients and benefits are often temporary due to disease progression. There are no established circulating biomarkers to monitor treatment response. Molecular heterogeneity between metastatic tumors within a patient (intra-tumor heterogeneity) and treatment- driven clonal evolution complicate choice and scheduling of treatments and limit duration of response. Circulating levels of cancer mutations in cell-free plasma DNA (ctDNA) strongly correlate with treatment response and disease progression in breast and colorectal cancer. The goal of this project is to assess the utility of a ctDNA-based blood test for treatment monitoring and as a minimally-invasive alternative to tumor re- biopsies for tumor genotyping in patients with metastatic melanoma. This goal will be pursued through analysis of serial plasma, tumor and germline samples from patients with metastatic melanoma from three cohorts: 1) a completed clinical trial of anti-PD1 immunotherapy; 2) prospective collection from the Stand Up To Cancer Genomically Enabled Medicine for Melanoma trial; and 3) a prospective observational study of patients at Mayo Clinic Arizona, treating with standard of care immunotherapy and molecularly targeted drugs. The specific aims of this proposal are 1) to assess clinical validating of ctDNA as a biomarker for monitoring treatment response; 2) to evaluate ctDNA sequencing as an alternative to tumor biopsies to guide treatment selection and 3) to identify genomic drivers of acquired resistance to immunotherapy. For Aim 1, patient- specific mutations will be identified using tumor exome sequencing, followed by targeted digital sequencing to quantify ctDNA. Changes in ctDNA levels during treatment will be compared with response and progression on imaging to assess clinical validity. For Aim 2, concordance of plasma and tumor samples will be assessed using exome sequencing of pre-treatment plasma DNA and comparison with tumor exome sequencing. For Aim 3, new genomic alterations arising at disease progression on immunotherapy will be identified using exome sequencing of post-treatment plasma DNA. Genomic drivers of acquired resistance to anti-PD1 inhibitors will be identified by evaluating molecular pathways recurrently altered at disease progression across the cohort. At completion of this project, it is anticipated that the extent to which ctDNA analysis is useful in melanoma for tumor monitoring, as an alternative to (and potential improvement) over tumor re-biopsies for patient selection and as an approach for investigating acquired resistance to immunotherapy, will be established.